Time-Lapse Confocal Imaging of Neuronal Dendritic Dynamics in Mouse Retinal Explants

Abstract

Source: Ing-Esteves, S. & Lefebvre, J. L. Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations. J. Vis. Exp. (2021)

This video demonstrates the use of confocal microscopy to visualize dendritic dynamics in live mouse retinal explants. It outlines the steps for stabilizing the retina under a confocal microscope, performing epifluorescence imaging to visualize starburst amacrine interneurons, and capturing time-lapse 3D images to monitor changes in amacrine dendritic morphology.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Time-lapse confocal imaging of live whole-mount retina preparations

1. Assemble the live-imaging incubation chamber for an upright confocal microscope, as seen in Figure 1.
   NOTE: For inverted confocal systems, flat-mounts are placed retinal ganglion cells (RGC) side down directly onto the glass bottom coverslip of the incubation chamber. Once the retinas make contact with the coverslip, they cannot be moved.
2. Fill the chamber with oxygenated artificial cerebrospinal fluid (aCSF) and turn on the pump and temperature controller (temperature 32-34 °C, flow rate 1 mL/min). Do not allow the temperature to rise above 34 °C.
3. To transfer the retinal flat-mount to the perfusion chamber, stop the pump and remove the aCSF that is in the chamber. Place the mixed cellulose ester (MCE) disc with the retinal flat-mount into the (empty) incubation chamber.
4. Place a sample weight onto the flat-mount; pre-wet the weight to break the surface tension. Refill the chamber with the warmed aCSF, and circulate aCSF at ~1 mL/min.
5. Position the nosepiece with the 25x water dipping objective (numerical aperture 0.95) into the imaging chamber. Screen for the labeled cells of interest using epifluorescent light (Figure 1C).
6. Adjust the imaging volume to capture dendritic features of interest.
   NOTE: This study captured the complete dendritic arbor at 1024 x 1024 pixels per frame, z-step 1 µm, and a frame rate of 2 min between each z-stack. Final image sizes are ~100 µm x 100 µm x 20 µm.
7. To adjust the laser power to an optimal setting, use a look-up table that identifies both oversaturated and undersaturated pixels. While scanning, adjust the laser power such that no pixels are oversaturated (i.e., at an intensity of 255 or above). Continue imaging as long as required or until there is a significant and detectable decline in fluorescent signal and an increase in noise (typically 2-4 h).
   NOTE: Reduced laser power is recommended as deconvolution algorithms work optimally when pixels are distributed over the full dynamic range. Pixel intensity should not exceed 254; empirical analyses of neurites revealed that pixel values below 170 are ideal for deconvolution. Fast scan speeds (400-600 Hz) with line averaging (2-3) are preferable to single, slower scans of the same total pixel dwell time. The area of prolonged imaging often photobleaches, but other explant sections remain viable. Multiple regions in a flat-mount can be imaged, each for 2-4 h, with a total incubation time of 6 hours. Imaging sessions beyond 6 h have not been systematically tested. Neurite degradation and blebbing are signs that the explant viability is declining.

Results

Figure 1: Live-cell imaging incubation chamber setup. (A) Live-imaging incubation apparatus sh...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Mixed cellulose ester membrane (MCE) filter papers, hydrophilic, 0.45 µm pore size	Millipore	HABG01 300
Polyethylene disposable transfer pipette	VWR	470225-034
Chamber polyethylene tubing, PE-160 10'	Warner Instruments	64-0755
Dual channel heater controller, Model TC-344C	Warner Instruments	64-2401
HC FLUOTAR L 25x/0.95 W VISIR dipping objective	Leica	15506374
Heater controller cable	Warner Instruments	CC-28
Large bath incubation chamber with slice support	Warner Instruments	RC-27L
MPII Mini-Peristaltic Pump	Harvard Apparatus	70-2027
PM-6D Magnetic Heated Platform (incubation chamber heater)	Warner Instruments	PM-6D
Pump Head Tubing Pieces For MPII Mini-Peristaltic Pump	Harvard Apparatus	55-4148
Sample anchor (Harps)	Warner Instruments	64-0260	Sample anchor must be compatible with incubation chamber
Sloflo In-line Solution Heater	Warner Instruments	SF-28
Calcium chloride dihydrate	Sigma-Aldrich	C7902
Carbogen (5% CO₂, 95% O₂)	AirGas	X02OX95C2003102	Supplier may vary depending on region
D-(+)-Glucose	Sigma-Aldrich	G7021
HEPES, Free Acid	Bio Basic	HB0264
Hydrochloric acid solution, 1 N	Sigma-Aldrich	H9892
Magnesium chloride hexahydrate	Sigma-Aldrich	M2670
pH-Test strips (6.0-7.7)	VWR	BDH35317.604
Potassium chloride (KCl)	Sigma-Aldrich	P9541
Sodium chloride (NaCl)	Bio Basic	DB0483
Sodium phosphate monobasic	Sigma-Aldrich	RDD007
ImageJ	National Institutes of Health (NIH)	Open source